Toxys is a Dutch biotech company that provides innovative in vitro toxicity screening solutions to rapidly identify hazardous and potential carcinogenic properties of novel compounds during the early phases of product and drug development.


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Toxys is developing the ReproTracker® assay, a state-of-the-art in vitro assay to visualize the key events during early embryonic development that allows us to reliably assess the potential developmental toxicity hazards of new drugs and products. This unique assay provides insight into the toxic mode-of-action.

Key Characteristics

  • Reporter assay for developmental toxicity
  • Visualisation of key developmental pathways
  • Insight into mode of action
  • Human stem cell based assay

Reporter based assay for developmental biology

Toxys is developing the ReproTracker® assay, a state-of-the-art in vitro assay to visualize the key events during early embryonic development that allows us to reliably assess the potential developmental toxicity hazards of new drugs and products. The assay is build on the technical and scientific background that has been developed by Toxys. We have combined our expertise on stem cell differentiation with the reporter technology that was applied in our ToxTracker assay and generated the ReproTracker reporter system that can identify developmental toxicity upon exposure to novel drugs, chemicals and materials. ReproTracker is able to identify compounds that interfere with embryonic development and provide insight into the underlying mechanisms of toxicity.

What is ReproTracker

The ReproTracker is a stem cell-based assay and uses fluorescent reporters to visualize the various stages of early embryonic development. Stem cells are generally accepted as an excellent platform for in vitro developmental toxicity hazard identification. ReproTracker consist of 6 different stem-cell lines (see below which reporter genes are used) each containing a different GFP reporters for the various crucial developmental pathways. These 6 biomarkers are choses as they represent specific and different phases of differentiation of stem cells. When the stem cells progresses in differentiation the various reporter genes will give a fluorescence signal when the specific reporter in that line is activated and a certain development stage has been reached. Should a compound interfere with the development the fluorescent signal will decrease. The response of the reporters to compounds is quantified using flow cytometry. The considerable advantage of using reporter cell lines is that it identifies which phase of the differentiation the compound disturbs. Therefore, the assay gives insight into the cellular pathways of the embryonic pathology. Additionally, when using different cell lines, it is possible to see in one assay if a chemical can induce multiple teratogenic mechanisms.

Unique biomarkers

Six different reporter cell lines forming a baseline profile for differentiation to cardiomyocytes and hepatocytes have been selected.


Octamer-binding transcription factor 4 (Oct4) is activated in pluripotent stem cells and works together with Sox2 and Nanog to suppress genes for differentiation and activate genes for self-renewal (Boyer et al., 2005). SRY-box17 (Sox17) and Forkhead box A2 (Foxa2) act in the endoderm state and can thus be indicators for hepatocyte differentiation. Sox17 stimulates the expression of Gata4 and Gata6, two genes known to be active in the extra-embryonic endoderm. It also inhibits the expression of the Sox2, Nanog and Oct4 (Niakan et al., 2010). However, Sox17 is also proven to be enriched in the cardiac mesoderm state (Liu et al., 2007). Foxa2 binds to the Wnt6 promoter and together with Gata6, it switches on the Wnt6 reporter construct, which subsequently activates the Wnt/β-catenin pathway. This results in the formation of the extra-embryonic endoderm (Hwang & Kelly, 2012). Bone morphogenetic protein 4 (Bmp4) is a member of the TGF-β family,  which plays a essential role in growth and differentiation. Studies showed that mutants in which Bmp4 is inactivated in mouse ES cells stop differentiating before the mesodermal stage and do not express the mesodermal marker Brachyury (Winnier, Blessing, Labosky, & Hogan, 1995). Vegfr stimulates differentiation to cardiomyocytes by activation of the ERK pathway, resulting in activation of the tyorsine kinase receptors Flk-1 and Flt-1. Flk-1 is a mesoderm marker, where cardiogenesis is initiated. Flt-1 is expressed in mature cardiomyocytes (Chen et al., 2006). CK18 is active in mature hepatocytes. CK18 is proven to bind to thrombin-antithrombin (TAT). TAT is, without the interaction with CK18, bound to hepatic receptors, inhibiting the hepatocyt differention. By removal of TAT, the cell can start to differentiate to hepatocytes (M. J. Wells et al., 1997).


We are currently completing the development of the 6 reporter cell lines. Once this has been finalized we will start a validation study with reference compounds.


The assay is currently under development, and therefore is not available for testing of novel compounds. Should you wish to stay updated on developments, or if you have reference compounds that you would like to test in the assays please contact us via the enquiries form below or by sending an email to

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